Project Summary/Abstract (30 lines) Tau protein stabilizes microtubules in neurons, but abnormal hyperphosphorylation of tau leads to aggregate formation. In addition, soluble tau intermediates are more neurotoxic than higher order aggregates, and are responsible for the cognitive dysfunction in AD. A beneficial AD treatment may be to enhance tau clearance. Mebendazole is an approved anthelmintic drug that binds to free tubulin. In a 14-day proof of concept study, we discovered that twice daily dosing of mebendazole (25 mg/kg, b.i.d.) significantly lowered tau protein levels in the cortex of 12 week old rTG4510 mice. Although mebendazole is used safely in humans, its poor solubility, metabolic stability, and pharmacokinetics, in particular for a CNS disease, limits its potential for translation to treat AD in human. Therefore, an exciting opportunity exists for medicinal chemistry optimization of both the PK and PD properties of novel benzimidazole analogs, which we believe will result in the discovery of a significantly improved molecule with the potential to treat AD. The specific aims to this proposal are: Aim 1. Medicinal chemistry optimization of tubulin binding, tau modulation and brain penetration of novel benzimidazoles. New analogs of mebendazole will be designed, synthesized, and characterized in the in vitro assays described in detail in the proposal. Chemistry approaches focus on increasing the solubility of new molecules and improving the stability of the vulnerable carbamate and ketone groups. Goals are to identify patentable novel chemical matter with improved in vitro drug-like properties and to determine the best compounds to advance to the Aim 2 pharmacokinetic and target engagement studies. Aim 2. Pharmacokinetics and target engagement studies of lead compounds in mice. The PK properties of compounds that satisfy the criteria 1-9 in Table 3 will be studied in mice, and those compounds with appropriate plasma and brain exposure will advance into in vivo studies to measure target engagement. The goal of the target engagement studies is to determine the effect of 28-day dosing of lead compounds on levels of total tau and p-tau in: (i) cerebrospinal fluid (CSF); and (ii) brain in the rTG4510 mouse model of AD. Aim 3. In vivo efficacy studies of the lead compounds in the rTG4510 mouse model. We will evaluate the efficacy of the lead compound in a longer, dose-response efficacy study in the rTG4510 mouse model of AD. Dosing will be determined from the PK and target engagement studies (Aim 2). We will study both: (i) preventative (early) treatment that will begin at 2 months, which is the time point of onset pathology, and continue for 3 months; and (ii) therapeutic (late) treatment that will start at 5 months, which is the start of cognitive decline ending at 8 months, when very severe pathology with cognitive deficits are exhibited. The goal is to identify a lead compound that gives: (i) a significant reduction in tau and p-tau leve...